Emergency Department Management of Radiation Casualties

A novel mycovirus that is related to the human pathogen Hepatitis E virus and 1

rubi-like viruses 2

Huiquan Liu1,2

, Yanping Fu2, Daohong Jiang

1,2﹡, Guoqing Li

1,2, Jun Xie

1,2, Youliang Peng

3, 3

Xianhong Yi2, Said A Ghabrial

4 4

1, National Key Laboratory of Agriculture Microbiology, Huazhong Agricultural University, Wuhan 5

430070, Hubei Province, P R China 6

2, The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and 7

Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei Province, P R China 8

3, Department of Plant Pathology, China Agricultural University, Yuanmingyuan West Road No. 1, 9

Haidian District, 100093, Beijing, P R China 10

4, Department of Plant Pathology, University of Kentucky, 201F Plant Science Building, 1405 11

Veterans Drive, University of Kentucky, Lexington, KY 40546-0312, USA 12

﹡ Corresponding author 13

Dr Daohong Jiang, Professor 14

Plant Pathology, College of Plant Science and Technology, Huazhong Agricultural University 15

Wuhan, 430070, Hubei Province, P R China 16

Tel: 86-27-87280487; Fax: 86-27-87397735; E-mail: [email protected] 17

﹟Current address: The college of Life Science, Hainan University, Haikou, Hainan Province, P R 18

China 19

Total number of words in text: 4266 20

Tables,2 and figures, 5 21

Summary: 287 words 22

Supplementary materials: Table, 1 and figures, 2 23

Running title: Mycovirus related to hepatitis E virus 24

GenBank Accession Number：EU779934 25

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.01897-08 JVI Accepts, published online ahead of print on 10 December 2008

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Abstract 26

Previously, we reported that three dsRNA segments, designated L, M and S, were detected in 27

Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M dsRNA segment was derived from the 28

genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum 29

debilitation-associated RNA virus. Here we present the complete nucleotide sequence of the L 30

dsRNA, which is 6043 nucleotides in length, excluding the poly (A) tail. Sequence analysis 31

revealed the presence of a single open reading frame (nt positions 42-5936) that encodes a protein 32

with significant similarity to the replicases of “alphavirus-like” supergroup of positive-strand RNA 33

viruses. Sequence comparison of the L dsRNA-encoded putative replicase protein containing 34

conserved methyltransferase, helicase and RNA-dependent RNA polymerase motifs showed that it 35

has significant sequence similarity to the replicase of Hepatitis E virus (HEV), a virus infecting 36

humans. Furthermore, we presented convincing evidence that the virus-like L-dsRNA could 37

replicate independently with slight impact on growth and virulence of its host. Our results suggest 38

that the L dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA 39

virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is 40

the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic 41

and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it 42

clustered with the rubi-like viruses, and that it is related to the plant clostero-, beny- and 43

tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related 44

alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings 45

suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might 46

have radiated horizontally among vertebrates, insects, plants and fungi. 47

KEY WORDS: Mycovirus, Sclerotinia sclerotiorum, Sclerotinia sclerotiorum RNA virus L, 48

Hypovirulence, Hepatitis E Virus, Virus evolution, Strain Ep-1PN, Sclerotinia sclerotiorum 49

debilitation-associated RNA virus 50

51

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Introduction 52

Sclerotinia sclerotiorum is a destructive soil borne plant pathogenic fungus with a wide host 53

range that includes more than 450 species and subspecies among 64 genera of plants (6). As an 54

important and unique plant pathogenic fungus, the sequence of the whole genomic DNA of S. 55

sclerotiorum has been determined 56

(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=fungi). DsRNA-associated 57

hypovirulence in S. sclerotiorum was first reported for strain 91 (5) and later for strain Ep-1PN (36). 58

In strain Ep-1PN, three dsRNA segments, designated L, M and S-dsRNAs with estimated sizes of 59

7.4, 6.4 and 1.0 kbp, respectively, were associated with hypovirulence of S. sclerotiorum (36). Of 60

these three dsRNA segments, only the M-dsRNA was consistently detected in association with the 61

hypovirulence phenotype (36). Evidence was recently presented that the M-dsRNA was derived 62

from the genomic RNA of a positive-strand RNA virus, Sclerotinia sclerotiorum 63

debilitation-associated RNA virus (SsDRV) (67). Furthermore, sequence analysis of the S-dsRNA 64

segment showed that it is a defective RNA derived from SsDRV. The L-dsRNA segment may 65

represent a novel mycovirus different from SsDRV since it lacks sequence similarity to SsDRV, as 66

determined by northern hybridization analysis (Daohong Jiang, unpublished data). 67

Discovery of novel mycoviruses may expand our knowledge of global ecology and evolution 68

of viruses. Although mycoviruses typically have isometric particles and dsRNA genomes (e.g., 69

members of the families Totiviridae, Chrysoviridae and Partitiviridae) (14), viruses in these 70

families also infect organisms other than fungi. Whereas some members in the family Totiviridae 71

infect protozoa, a number of the viruses in the families Partitiviridae and Chrysoviridae infect 72

plants. The mycoreoviruses from hypovirulent strains of Cryphonectria parasitica and Rosellinia 73

necatrix represent a distinct group of dsRNA mycoviruses with reovirus-like particle morphology, 74

and they are most closely related to the tick-borne animal pathogens belonging to the genus 75

Coltivirus in the family Reoviridae (19, 52). Viruses with dsRNA genomes infect a broad range of 76

hosts (vertebrates, invertebrates, fungi, plants, protozoa, and bacteria) and are grouped in six 77

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families of dsRNA viruses: Totiviridae, Birnaviridae, Partitiviridae, Cystoviridae, Chrysoviridae, 78

and Reoviridae (10). Comparative analysis of the amino acid sequences of proteins encoded by 79

dsRNA viruses revealed little similarity between viruses of different genera, even those belonging 80

to the same family, e.g. those belonging to the family Reoviridae. Even though the RdRps are the 81

most highly conserved genes among RNA viruses, phylogenetic analysis of the RdRps suggests a 82

polyphyletic origin for dsRNA viruses. The dsRNA viral RdRps tend to group with different 83

supergroups of the positive-strand RNA viruses (29). The concept of multiple origins of dsRNA 84

viruses from diverse lineages of positive-strand RNA viruses is presently well accepted (1, 29). 85

Recently, there is an increasing number of reports of positive-strand RNA mycoviruses whose 86

RdRp and helicase gene lineages are within the lineages of positive-strand RNA plant viruses: e.g., 87

the potexvirus-like mycoviruses FgV-DK21 in Fusarium graminearum (33), Botrytis virus X (22) 88

and Oyster mushroom spherical virus (65). Many of these positive-strand RNA mycoviruses do not 89

encode coat proteins and they occur in their hosts as dsRNA derivatives of their genomic 90

positive-strand RNAs, but are phylogenetically related to plant viruses. The mycoviruses that lack 91

typical virions include: members of the genus hypovirus that infect Cryphonectria parasitica, with 92

lineage to plant potyviruses (44); SsDRV, an unassigned mycovirus from Sclerotinia sclerotiorum 93

(67), which is related to allexiviruses in the family Flexiviridae, and Diaporthe ambigua RNA virus 94

(DaRV), with lineage to tombusviruses (47). Mitoviruses that infect C. parasitica (46), 95

Ophiostoma novo-ulmi (20) and Botrytis cinerea (66) are phylogenetically related to positive-strand 96

RNA bacteriophages in the family Leviviridae. Considering the fact that these related 97

positive-strand RNA viruses infect a wide variety of organisms, the ancestral RNA virus might be of 98

ancient origin and/or might have spread out horizontally among animals, plants, fungi, protozoa and 99

prokaryotes. 100

Some mycoviruses are associated with debilitation/hypovirulence of their hosts, and these 101

mycoviruses are potential bio-control agents to combat plant fungal diseases and to probe the 102

pathogenicity of host on molecular level (44). Among the debilitation/hypovirulence-associated 103

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mycoviruses, the hypovirus/C. parasitica system has been the most thoroughly studied. Significant 104

insight has been gained on the molecular basis of hypovirulence in this system and its potential 105

implementation for biological control of chestnut blight (27, 38, 44, 55). The depth of knowledge 106

gained from studying the hypovirus/C. parasitica system should now pave the way for 107

investigations on other similar fungal virus systems. 108

In a recent study, Li et al. (37) identified a small number of genes whose expression was down 109

regulated in the virus-infected S. sclerotiorum strain Ep-1PN and discussed the probability that the 110

predicted depleted levels of the corresponding proteins may contribute to the characteristic 111

debilitation and hypovirulence of this strain. In the present study, molecular cloning and 112

sequencing of the L-dsRNA segment from a debilitated fungal strain were carried out and the 113

sequences generated were assembled and subjected to sequence and phylogenetic analyses to 114

determine whether the L-dsRNA is related to previously characterized mycoviruses and to examine 115

its relationships to viruses infecting organisms other than fungi. 116

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Methods and materials 117

Fungal Strains 118

S. sclerotiorum hypovirulent strain Ep-1PN, which contained L-dsRNA, M-dsRNA (SsDRV), 119

and S-dsRNA, was originally isolated from a sclerotium collected from a diseased eggplant (35). 120

Strain Ep-1PNA367, a virus-free strain, was a single-ascospore isolate derived from Ep-1PN. Strain 121

Ep-1PNSA-8, Ep-1PNSA-23 and Ep-1PNSA-34 were isolated from individual sclerotia of strain 122

Ep-1PN. All fungal strains were grown at 18–22 oC on potato dextrose agar medium (PDA) and 123

stored on PDA slants at 4–8 oC. 124

Extraction of double-stranded RNA 125

The procedure for dsRNA extraction, previously described by De Paulo and Powell (7), was 126

used with minor modifications. For the extraction of dsRNA from mycelium of strains Ep-1PNSA-8, 127

Ep-1PNSA-23, Ep-1PNSA-34 and Ep-1PNA367, small agar mycelial plugs were placed on 128

cellophane membranes placed on top of the PDA medium (CM-PDA) in Petri plates for 2 days, and 129

then mycelium was harvested from the cellophane membranes. To extract the dsRNA from strain 130

Ep-1PN, the mycelium growing on CM-PDA for up to one week was harvested, and then 131

homogenized in a sterilized mortar with a pestle. The homogenate was spread on fresh CM-PDA 132

plates with cellophane membranes for 2 days, and then mycelium was harvested and stored at -80 133

oC. 134

cDNA synthesis, molecular cloning and sequencing 135

To obtain sequence information for the L-dsRNA, dsRNA (1.0 µg) was mixed with 0.1 µg 136

random hexamer primers and 3 µl 100% DMSO, and DEPC-treated ddH2O was added to a final 137

volume of 12 µl. The mixture was heated at 95–98 oC for 14 min and chilled on ice for 3 min. 138

First and second-strand cDNAs were synthesized as described by Sambrook et al. (51). The 139

resulting cDNA was purified by filtration through a Sephadex G-50 column and A-tailed with Taq 140

DNA polymerase and dNTP at 72 oC for 30 min. The A-tailed double stranded cDNA was ligated 141

into the pMD18-T vector according to the manufacturer’s instructions (TaKaRa) and transformed 142

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into competent cells of E. coli JM109. Sequence-specific primers were used for RT-PCR to amplify 143

parts of the genome which were not cloned by the initial random cDNA synthesis. Denatured 144

dsRNA was reverse transcribed using RevertAidTM

M-MuLV Reverse Transcriptase (Fermentas) 145

and a sequence-specific reverse primer and incubated for 60 min at 45 o

C. After reverse 146

transcription, the mixture was treated with RNase H (1 U at 37 oC for 30 min; TaKaRa) and 2 % of 147

the reaction volume was used for PCR amplification with the pertinent forward and reverse primers, 148

GC Buffer and LA Taq DNA Polymerase (TaKaRa). The resulting PCR product was fractionated by 149

electrophoresis on 1% agarose gel and purified using a gel extraction kit (Axygen). The PCR 150

product was cloned into the pMD18-T vector. 151

Clones for the terminal sequences of the dsRNA were generated by T4 RNA ligase 152

oligonucleotide-mediated amplification as described by Lambden et al. (34). The 3’ terminus of 153

each strand of dsRNA was ligated at 5–15 o

C for 16–18 h with the 5’-end phosphorylated 154

oligonucleotide 5’-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC- 155

(NH2)-3’ using T4 RNA ligase (Fermentas). The oligo-nucleotide-ligated dsRNA was denatured 156

and used for the reverse transcription reaction with RevertAidTM

M-MuLV Reverse Transcriptase 157

and 10 pmol of a primer with sequence complementary to the oligonucleotide used for the RNA 158

ligation (oligoREV, 5’-GGCAATTAACCCTCACTAAAG-3’). The reaction product was treated 159

with RNase H, as described above, and the cDNA was amplified with another primer 160

complementary to the RNA ligation oligonucleotide (5’-TCACTAAAGAATTCGATCGATC-3’) 161

and the sequence-specific primer corresponding to the 5’- and 3’-terminal sequences of the dsRNA, 162

respectively. The expected PCR products were recovered and purified with a gel extraction kit 163

(Axygen), cloned into the pMD18-T vector (TaKaRa). 164

Sequencing was carried out by the dideoxynucleotide termination method using a Big Dye 165

Terminator Sequencing kit (BigDye terminator v. 2.0; ABI) and an ABI PRISM 377-96 automated 166

sequencer (Beijing Sunbiotech). M13 universal primers or sequence-specific primers were used for 167

sequencing and each base was determined by sequencing at least two independent clones (usually 168

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three to five clones) from both orientations. 169

Sequence and phylogenetic analyses 170

The DNAMAN 5.2.9 version (Lynnon Biosoft, USA) software package was used for sequence 171

annotations, including nucleotide statistics and open reading frame (ORF) searching. Sequence 172

similarity searches of GenBank, Swissprot and EMBL databases were conducted using the BLAST 173

program (3). Searches for amino acid signatures and protein motifs were conducted using the 174

programs included in the ExPASy proteomics tools (http://www.expasy.org/tools/). Multiple 175

alignments of amino acid were made with the program MUSCLE Version 3.6 (9) and the resulting 176

alignment was manually adjusted according to Koonin’s alignments (28). Two independent methods 177

for the generation of tentative phylogenetic trees were used, namely Neighbor-joining (NJ) 178

algorithm and Maximum likelihood (ML) method. Neighbor-joining (NJ) algorithm was performed 179

using PAUP* 4.0b10 (59), assuming the BLOSUM 62 matrix (18). Bootstrap values were 180

calculated from 1000 bootstrap replicates. Maximum likelihood (ML) method was performed 181

using program TREE-PUZZLE version 5.2 (54). Likelihoods were calculated using the VT model 182

of amino acid substitution (42) and the relevant parameter values estimated from the data (available 183

upon request). 184

Northern blot hybridization 185

Northern hybridization analysis was performed as previously described (25). To verify the 186

authenticity of the cDNA clones generated with the purified dsRNA, the cDNA clones were labeled 187

with [32

P] dCTP using a radio-labeling kit (TaKaRa) and used to probe the RNA blot. 188

Reverse transcription-PCR 189

Total RNA from isolates derived from the debilitated strain Ep-1PN was isolated according to 190

Sambrook et al. (51). First-strand cDNA was synthesized using RevertAidTM

M-MuLV Reverse 191

Transcriptase (Fermentas). To detect M-dsRNA, the reverse primer SsDRV-PCRpREV 192

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(5’-CAGTCCCTAGTTTCATCTCGTTCC-3’) was used and the first-strand cDNA was then 193

subjected to PCR using the SsDRV-PCRpREV primer and the forward primer SsDRV-PCRpFOR 194

(5’-TGCAGGAAACAGTCATGGCAAC-3’) with a predicted size of 871 bp. To detect L-dsRNA, 195

the reverse primer Ss-7.4RP (5’-GAAGCCACAGGGACAGCAAG-3’) was used and the 196

first-strand cDNA was then subjected to PCR using the Ss-7.4RP primer and the forward primer 197

Ss7.4-FP (5’-CCACCGACGCAGGCAAATAC-3’) with a predicted size of 721 bp. The conditions 198

for cDNA amplification included an initial denaturation step of 4 min at 94℃, followed by 30 199

cycles of 30 sec at 94℃, 30 sec at 61℃ and 1 min at 72℃, with a final elongation step of 10 min at 200

72℃. PCR products were fractionated by gel electrophoresis on 1% agarose gels and stained with 201

ethidium bromide. 202

Mycelial growth and virulence test 203

To evaluate the effect of SsRV-L on colony morphology and virulence on rapeseed of 204

Sclerotinia sclerotiorum, SsRV-L-infected strains were compared with the original strain Ep-1PN 205

and virus-free ascospore offspring of strain Ep-1PN using the procedures of Li et al. (35). 206

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Results 207

Synthesis and sequencing of cDNA from L-dsRNA 208

The L-dsRNA segment extracted from mycelia of strain Ep-1PN was electrophoretically 209

separated on 1% agarose gel and purified with a gel extraction kit, and then, subjected to cDNA 210

synthesis using random primers (hexamer). The ds-cDNA fragments were cloned and the cloned 211

cDNAs were transformed into E. coli strain JM109. More than forty cDNA clones with inserts of 212

200 to 800 bp were obtained and confirmed to be derived from the L-dsRNA using Reverse 213

Northern dot-blot hybridization analysis (data not shown). Among these, 10 randomly selected 214

cDNA clones were sequenced in both directions (Fig. 1A; clones A to J). RT-PCR was conducted to 215

fill the gaps between clones with specific primers designed based on these cDNA sequences, and 216

used an oligo-ligation strategy to obtain the sequences of the 5' and 3' termini. A total of 26 clones 217

were obtained and sequenced (Fig. 1A). Computer-assisted sequence assembly showed that the 218

full-length L-dsRNA cDNA is 6043 bp in length, excluding the poly(A) tail. The cloning strategy 219

for dsRNA is outlined in Fig. 1A. The sequence was deposited in the GenBank under accession no. 220

EU779934. 221

Sequence analysis of the complete cDNA of the L-dsRNA 222

Analysis of the complete cDNA sequence revealed the presence of a single large open reading 223

frame (ORF; nt 42–5936), potentially encoding a polypeptide of 1964 amino acid residues with a 224

predicted mass of 213.4 kDa (Fig. 1B). Motif Scan searches showed that this protein contains 225

conserved methyltransferase, viral RNA helicase and RNA-dependent RNA polymerase (RdRp) 226

domains characteristic of the replicases of many of the positive-strand RNA viruses in the 227

alphavirus-like supergroup of viruses (Fig. 1B). The 5’ noncoding region consists of 41 nucleotides 228

and could be folded into a hairpin structure, and the 3’ noncoding region consists of 107 nucleotides 229

and could be folded into three independent hairpin structures (see Fig. S1 in the supplemental 230

material). Such hairpin structures are also found in other viruses in the alphavirus-like supergroup 231

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(15, 17, 41, 43, 58). Homology searches of the methyltransferase, helicase and RdRp conserved 232

motifs of the L-dsRNA-encoded replicase indicated that they are related to several animal viruses 233

belonging to the genus Hepevirus including Hepatitis E virus (HEV), swine hepatitis E virus 234

(HEV-swine) and Avian hepatitis E virus (AHEV) (Fig. 2). BLASTP database searches of the 235

L-dsRNA RdRp conserved domain revealed that it shares significant sequence similarity (E values 236

of 3e-11

or lower) with the RdRp encoded by the hepeviruses and by the closteroviruses. The RdRp 237

domain of L-dsRNA shares 27% identity and 43% similarity with HEV, and 28% identity and 44% 238

similarity with HEV-swine (Table 2). These L-dsRNA RdRp conserved motifs are also related to 239

those of plant viruses in the genus Closterovirus. The RdRp conserved motifs of SsRV-L share 23 % 240

identity and 40 % similarity with mint virus 1. Likewise, the identity and similarity scores 241

between SsRV-L and plum bark necrosis and stem pitting-associated virus are 24 % and 41 %, 242

respectively. The corresponding scores for mint vein banding virus are 24 % and 40 % (Table 2). 243

Thus, these results suggest that the L-dsRNA probably represents the replicative form or a 244

replicative intermediate of the genomic RNA of a positive-strand mycovirus co-infecting the 245

debilitated S. sclerotiorum strain Ep-1PN along with SsDRV. The newly characterized virus 246

co-infecting strain Ep-1PN was designated Sclerotinia sclerotiorum RNA virus-L (SsRV-L). 247

Sequence comparison of the viral RNA helicase motifs of SsRV-L showed that they share 248

significant sequence similarity with viruses in the genera Hepevirus and Omegatetravirus (E values 249

of 4e-6

or lower). The helicase motifs identity and similarity scores between SsRV-L and AHEV or 250

HEV are 28 % and 41 %, or 29 % and 42 %, respectively. Interestingly, the RNA helicase motifs of 251

SsRV-L also share significant sequence similarities with the insect viruses Helicoverpa armigera 252

stunt virus (HaSV) and Dendrolimus punctatus tetravirus (DpTV), the percent identity and 253

similarity of the helicase domain between SsRV-L and those two insect viruses are 27 % and 41 %, 254

respectively (Table 2). Both HaSV and DpTV belong to the genus Omegatetravirus in the family 255

Tetraviridae (15, 64). 256

Furthermore, the methyltransferase motifs of SsDRV-L and AHEV share sequence similarities 257

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with the tobamoviruses tomato mosaic virus, tobacco mosaic virus, pepper mild mottle virus, 258

cucumber green mottle mosaic virus and rehmannia mosaic virus, and the insect betatetravirus 259

Nudaurelia capensis beta virus (Table 2). 260

Phylogenetic analysis 261

Maximum likelihood distance comparisons of amino acid sequences of the RdRp domain of 262

SsRV-L and representative viruses of alphaviruses, endornaviruses, tymo-like viruses, rubi-like 263

viruses and tobamo-like viruses showed that SsRV-L is most closely related to Hepatitis E virus 264

belonging to the genus Hepevirus (see Table S1 in the supplemental material). Phylogenetic trees 265

based on multiple alignments of RdRp conserved motifs of SsRV-L and these viruses were 266

independently generated with Neighbor-joining (NJ) algorithm and Maximum likelihood (ML). The 267

resulting NJ and ML trees had similar topology and showed that SsRV-L clusters with several 268

rubi-like viruses including benyviruses, hepeviruses, omegatetravirus and rubivirus. The ML tree 269

is shown in Fig. 3A. 270

A phylogram based on multiple alignments of viral RNA helicase conserved motifs of SsRV-L 271

and representative alphaviruses, endornaviruses, tymo-like viruses, rubi-like viruses and 272

tobamo-like viruses was similar to the tree generated by multiple alignments of RdRp conserved 273

motifs (Fig. 3B). Likewise, a similar tree was generated based on the multiple alignments of 274

methyltransferase conserved motifs of SsRV-L and selected viruses (see Fig. S2 in the supplemental 275

material). 276

Evidence for autonomous replication of SsRV-L 277

The single-sclerotium isolates Ep-1PNSA-8, Ep-1PNSA28 and Ep-1PNSA32, which were 278

derived from strain Ep-1PN, lacked the M-dsRNA and S-dsRNA segments, but contained only the 279

L-dsRNA segment. This was confirmed with RT-PCR detection (Fig. 4). Thus, SsRV-L could 280

replicate independently in S. sclerotiorum. 281

Compared to the virus-free strain Ep-1PNA367, an ascospore isolate derived from strain 282

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Ep-1PN, the hyphal growth and virulence on detached rapeseed leaves of strains Ep-1PNSA-8, 283

Ep-1PNSA28 and Ep-1PNSA32, were slightly reduced, but their growth and virulence were 284

significantly higher than those of the hypovirulent strain Ep-1PN (Fig. 5). The sclerotial growth was 285

normal on the PDA medium and there was little or no difference in colony morphology between 286

these strains and that of the wild type strain of S. sclerotiorum. Thus, SsRV-L contributes little if 287

any to the debilitation of strain Ep-1PN. 288

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Discussion 289

This study represents the first report on molecular characterization of a positive strand RNA 290

mycovirus, SsRV-L that is phylogenetically related to the human pathogen, Hepatitis E virus (HEV) 291

and rubi-like viruses. Sequence analysis of the full-length cDNA clone containing the coding 292

sequence of a putative viral replicase revealed the presence of conserved methyltransferase, viral 293

RNA helicase and RdRp-2 superfamily motifs characteristic of the viral RNA replicase proteins of 294

positive-strand RNA viruses and with significant similarity to the human pathogen HEV, a member 295

of the genus Hepevirus (30). Like HEV, the genome of SsRV-L also has a short 5’-untranslated 296

leader sequence and 3’ untranslated region with poly(A) tail. However, unlike the genomes of 297

HEV and HEV-swine, which contain three ORFs, SsRV-L genomic RNA contains only a single 298

ORF (ORF1) with significant sequence similarity to ORF1 of HEV and those of HEV-swine and 299

AHEV. Furthermore, ORF1 of SsRV-L lacks coding sequences for papain peptidase or papain 300

peptidase-like proteins. Interestingly, the viral RNA helicase and methyltransferase motifs of 301

SsRV-L and HEV (including HEV-swine and AHEV) share significant sequence similarity with the 302

insect tetraviruses and the plant tobamo-like viruses (Table 2). 303

The positive strand RNA viruses were classified into three superfamilies: alpha-like, 304

picorna-like and flavi-like. The superfamily of alpha-like viruses comprises three lineages: rubi-like, 305

tobamo-like and tymo-like viruses (31). Whereas tobamo- and tymo-like viruses infect plants, the 306

rubi-like viruses infect plants, vertebrates and insects. Recently, evidence was presented that the 307

benyvirus (rubi-like lineage) Beet necrotic yellow vein virus (BNYVV) replicates in its 308

plasmodiophorid vector Polymyxa betae suggesting that BNYVV may also be considered a virus of 309

plasmodiophorids (39). Our results from phylogenetic analysis support the conclusion that 310

SsRV-L could be classified with the rubi-like viruses, thus, the host range of rubi-like viruses is 311

more diverse than once thought. The reason for the broad host range of rubi-like viruses is not 312

clearly known. 313

Although, SsRV-L shares sequence similarities with HEV and the insect viruses HaSV and 314

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DpTV, it is more likely that theses positive-strand RNA viruses were derived independently from 315

plant viruses, possibly those related to tobamoviruses. Since vertebrates and insects, are not hosts of 316

S. sclerotiorum, it is unlikely that S. sclerotiorum acquired the ancestral SsRV-L from these 317

organisms. S. sclerotiorum, a plant pathogenic fungus, might have acquired the ancestral 318

SsRV-L-like virus from a virus-infected plant since it shares many hosts with viruses in the genus 319

Tobamovirus. As a matter of fact, the hypovirulent strain Ep-1PN was originally isolated from a 320

diseased plant of eggplant (Solanum melongena) (35). Humans might have obtained HEV from 321

herbivores, e.g., swine was one of the reservoirs of HEV (11), and wild animals, which could be 322

infected by HEV or HEV-like viruses (57). Insects, like Helicoverpa armigera and Dendrolimus 323

punctatus, could have obtained HaSV and DpTV from plants, and it is feasible that birds obtained 324

avian HEV from virus-infected insects. Although these inferences are consistent with the hypothesis 325

that the ancestral alpha-like positive-strand RNA virus might have spread out horizontally among 326

plants, vertebrates and insects, this does not rule out the ancient origin of the progenitor virus in a 327

single cell type prior to the separation of fungi, plants and animals. Recently, Koonin et al. (32) 328

presented evidence that picorna-like virus evolution antedates the radiation of eukaryotic 329

supergroups. 330

The genomic organization for SsRV-L and SsDRV are similar to each other, both of them lack 331

unnecessary genes including genes for coat protein and movement protein. Hypoviruses and DaRV 332

are also examples of mycoviruses that lack coat and movement proteins. These viruses, like SsRV-L 333

and SsDRV, are phylogenetically related to plant viruses. Botrytis virus F (BVF), represents an 334

example of a mycovirus that is phylogenetically related to plant viruses (potexviruses) but which 335

codes for a coat protein in addition to the replicase (21). Unlike potexviruses, however, BVF does 336

not code for a movement protein. It is not known how viruses delete unnecessary genes from their 337

genomes; SsDRV, SsRV-L, DaRV, hypoviruses and similar viruses may represent examples of 338

regressive evolution by viruses in fungi. 339

The occurrence of SsRV-L and SsDRV in the hypovirulent strain Ep-1PN of S. sclerotiorum 340

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represents a novel type of co-infection involving two positive strand RNA mycoviruses. Mixed 341

infections with two or more dsRNA viruses appear to be of common occurrence among 342

mycoviruses (14). Examples of mixed infections with dsRNA viruses belonging to distinct virus 343

families include the totivirus Hv190SV and the chrysovirus Hv145SV that co-infect C. victoriae (12, 344

13). There are many examples of mixed infections by distinct members of the same virus family 345

including the two totiviruses, SsRV-1 and SsRV-2 that co-infect Sphaeropsis sapinea (48), the two 346

totiviruses ScV-L-A and ScV-L-BC that infect Saccharomyces cerevisae and the partitiviruses 347

Penicillium stoloniferum viruses S and F that infect Penicillium stoloniferum. 348

As previously reported, it is not clear whether SsRV-L contributes to the hypovirulence 349

phenotype of strain Ep-1PN (36) since S. sclerotiorum isolates that carry SsRV-L alone exhibit a 350

normal phenotype. Thus, we assume that S. sclerotiorum might have acquired SsRV-L earlier than 351

SsDRV, and SsDRV could have been acquired through hyphal anastomosis with other 352

SsDRV-infected fungal strains since mycoviruses could be transmitted between vegetatively 353

incompatible strains though at lower frequencies. 354

Unlike C. parasitica that can produce both ascospores and conidial spores, S. sclerotiorum can 355

only produce ascospores that form in apothecia germinating from dormant sclerotia. Thus SsRV-L 356

and SsDRV cannot be dispersed via conidial spores. Furthermore, our previous work showed that 357

both SsRV-L and SsDRV could not be transmitted through ascospores of strain Ep-1PN (26, 67), 358

and hyphae do not present valid dormant material for dispersal of S. sclerotiorum. Therefore, 359

SsRV-L and SsDRV could only be transmitted and dispersed by sclerotia, and the distribution of 360

SsRV-L and SsDRV would then be confined to limited areas. Moreover, the survival ability of 361

fungal strains doubly infected with SsRV-L and SsDRV is predicted to be very low since they grow 362

slowly, virtually lose their virulence and produce only few sclerotia (35). Thus, we reasoned that 363

horizontal transfer of the ancestral viruses of SsRV-L and SsDRV from other fungi or plants to S. 364

sclerotiorum might have occurred relatively recently. Because S. sclerotiorum has a broad host 365

range of plants known to be susceptible to many closteroviruses and potexviruses, the possibility 366

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that closterovirus-like (SsRV-L) and potexvirus-like (SsDRV) viruses might have transferred from 367

plants to S. sclerotiorum seems feasible (8, 40). 368

SsRV-L appears to be more stable than SsDRV in S. sclerotiorum since subcultures that 369

contain SsRV-L but lacks SsDRV are relatively easy to obtain by hyphal tipping and single 370

sclerotium isolation. Like SsDRV, SsRV-L is also eliminated through sexual reproduction of S. 371

sclerotiorum; this phenomenon is common among mycoviruses of higher ascomycetous hosts, e.g., 372

the hypovirus/C. parasitica system (4), but the underlying mechanism is not known. 373

Currently, it is not understood whether there is an interaction between SsRV-L and SsDRV in 374

doubly infected S. sclerotiorum and whether this interaction has any bearing on the debilitation 375

phenotype since the two viruses can replicate independently. S. sclerotiorum strains singly-infected 376

with SsRV-L show little or no adverse effects, whereas strains that are singly infected with SsDRV 377

exhibit a debilitated phenotype. In a recent study, Li et al. (37) identified several genes whose 378

expression was down regulated in a doubly infected S. sclerotiorum strain. The 379

hypovirulence/debilitation system of Sclerotinia sclerotiorum and its associated mycoviruses 380

presents an attractive system to explore the molecular basis of pathogenicity in this devastating 381

plant pathogen (37). Future construction of infectious full-length cDNA clones for these two viruses 382

and the development of RNA transfection systems would be useful in deciphering the interaction 383

between SsRV-L and SsDRV-1 and their effects on their common host. 384 ACCEPTED

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Acknowledgment 385

This work was supported by grants from the National Basic Research Program (2006CB101901-1), 386

the Program for New Century Excellent Talents in University (NCET-06-0665) and the Fok Ying 387

Tong Education Foundation for Young Teacher of Universities and Colleges (No. 80125), and 388

our publication is in the memory of Mr Fok Ying Tong. We also thank the anonymous reviewers for 389

their constructive and helpful comments. 390

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Figure Legends 583

Figure 1. Schematic representation of the strategy used in cDNA cloning of SsRV-L dsRNA and 584

predicted genome organization. A: cDNA clones (A to J; red lines) were synthesized using random 585

hexamer primers and denatured dsRNA as a template. Sequences of the regions of the dsRNA that 586

were not covered by these cDNA clones were obtained from cloned RT-PCR products using 587

sequence-specific primers (designed based on the sequences of these cDNA clones), and clones 588

correspondent to 5’ and 3’ termini were amplified using the method of Lambden et al. (34) (Clones 589

1 to 26; black lines). B: Diagrammatic representation of the genomic organization of SsRV-L 590

dsRNA showing the presence of a single ORF. The ORF encodes a putative protein containing a 591

methyltransferase domain, a helicase domain typical of superfamily 1 of viral RNA helicases and 8 592

conserved motifs characteristic of RdRps of positive-strand RNA viruses. 593

594

Figure 2. Amino acid sequence alignment of the putative methyltransferase (A), helicase (B) and 595

RdRp (C) motifs of SsRV-L and those of selected viruses in the genus of Hepevirus. The positions 596

of the conserved motifs in these motifs (shaded areas) correspond to those previously described (28, 597

31, 49) and are indicated with horizontal lines above the shaded areas. Asterisks indicate identical 598

amino acid residues, and colons indicate similar residues. Numbers in brackets refer to the amino 599

acid position in the ORF. See Table 1 for abbreviations of virus names and viral protein accession 600

numbers. 601

602

Figure 3. Phylogenetic analysis of the conserved motifs and flanking sequences of RdRp (A) and 603

viral RNA helicase (B) derived from aligned deduced amino acid sequences of SsRV-L and selected 604

viruses. Neighbor-joining (NJ) algorithm and Maximum likelihood (ML) were used to generate 605

tentative phylogenetic trees independently. NJ algorithm was performed using PAUP* 4.0b10 (59) 606

and ML was performed using program TREE-PUZZLE version 5.2 (54). The resulting ML-tree is 607

shown. The first number indicated at the nodes represents the bootstrap values (%) calculated from 608

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the NJ tree inferred from 1000 bootstrap replicates and the second number represents the quartet 609

puzzling support values (%) inferred from 10000 puzzling steps; a minus sign (–) indicates that a 610

node is absent in the corresponding NJ method. Bootstrap or quartet puzzling support values of only 611

>50% are indicated. The scale relates branch lengths to the number of substitutions per site. The 612

tree was outgroup-rooted to the viruses in carmo-like group for RdRp analysis and to the arteri-like 613

viruses (IBV) for viral RNA helicase analysis. See Table 1 for abbreviations of virus names, viral 614

protein accession numbers and the amino acid positions in the replicase sequences of the viruses 615

used for phylogenetic analysis. 616

617

Figure 4 Northern hybridization analysis of Sclerotinia sclerotiorum strains singly or doubly 618

infected with Sclerotinia sclerotiorum RNA virus L (SsRV-L). Five strains of Sclerotinia 619

sclerotiorum were used: strain Ep-1PN is doubly infected with SsRV-L and SsDRV; strains 620

Ep-1PNSA-8, Ep-1PNSA-23 and Ep-1PNSA-34 are single-sclerotium-isolates derived from strain 621

Ep-1PN and contain only SsRV-L; and strain Ep-1PNA367, single-ascospore-isolate derived from 622

strain Ep-1PN and is virus-free,. Panel A, electrophoretic analysis of dsRNA samples on agarose 623

gels; Panel B, dsRNA samples were hybridized with an α-32

P labeled cDNA probe of SsRV-L; Panel 624

C, total RNA samples were reversely transcribed with MLV-reverse transcriptase and specific RT 625

primer (5’-CAGTCCCTAGTTT CATCTCGTTCC-3’) designed based on the sequence of SsDRV, 626

and PCR-amplified with SsDRV specific primers (Reverse 627

primer:5’-CAGTCCCTAGTTTCATCTCGTTCC-3’ and Forward Primer 628

5’-TGCAGGAAACAGTCATGGCAAC-3’) . 629

630

Figure 5 Effect of Sclerotinia sclerotiorum RNA virus L (SsRV-L) on hyphal growth and virulence 631

of its host. Five strains of Sclerotinia sclerotiorum were used: strain Ep-1PN is doubly infected with 632

SsRV-L and SsDRV; strains Ep-1PNSA-8, Ep-1PNSA-23 and Ep-1PNSA-34 are 633

single-sclerotium-isolates derived from strain Ep-1PN and contain only SsRV-L; and strain 634

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Ep-1PNA367, single-ascospore-isolate derived from strain Ep-1PN and is virus-free. Panel A，635

Comparison among five selected strains of S. sclerotiorum for their mycelial growth rate on PDA 636

plate at 20 oC; Panel B, Comparison among the five strains for their virulence on detached leaves 637

of rapeseed (Brassica napus) as determined by induced lesion diameter at 20 oC for 48 h. 638

639

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Figure 1

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Figure 2

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Figure 3A

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Figure 3B

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Figure 4

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Figure 5

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Table 1 Viruses selected for phylogenetic analysis 1

positions for phylo-

genetic analysis (aa) Family/Genus Species Abbreviation Accession no.

RdRp helicase

Hepevirus

Hepatitis E virus

Yam67

HEV-Yam67 AAM66329 1361-1616

Swine hepatitis E virus HEV-swine ABB88699 1373-1628 969-1199

Avian hepatitis E

virus*

AHEV AAS45830 1201-1457 800-1029

Hepatitis E virus JRA1 HEV-JRA1 BAB93536 971-1201

Closteroviridae

Closterovirus Beet yellows virus BYV AAC25115 2701-2963 2231-2519

YP_224091 90-354 Mint virus 1* MV1

YP_224291 1575-1857

YP_001552324 170-434 Ampelovirus Plum bark necrosis and

stem pitting-associated

virus*

PBNSTaV

YP_001552323 2029-2312

AAP87784 176-440 Little cherry virus 2 LChV-2

AAP87783 1327-1609

Unassigned Mint vein banding

virus*

MVBV AAS57939 171-435

Tobamovirus Tobacco mosaic virus TMV AAD44327 1285-1548 815-1087

Pepper mild mottle

virus

PMMoV CAC59955 1285-1548 815-1087

Rehmannia mosaic

virus*

ReMV YP_001041889 1285-1548 815-1087

Tetraviridae

Omegatetravirus Helicoverpa armigera

stunt virus

HaSV NP_049235 1030-1288 593-851

Dendrolimus punctatus

tetravirus*

DpTV YP_025094 1021-1279 584-842

Betatetravirus Nudaurelia capensis

beta virus

NβV NP_048059 981-1232 548-805

Togaviridae

Alphavirus Sindbis virus SINV NP_740669 274-537

NP_062888 708-969

Semliki forest virus SFV NP_740668 269-532

NP_463457 705-963

Aura virus AURAV NP_819013 268-531

Rubivirus Rubella virus RUBV NP_062883 1775-2035 1333-1578

Endornavirus

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Oryza sativa

endornavirus

OsEV YP_438200 4219-4482 793-1055

Oryza rufipogon

endornavirus

OrEV YP_438202 4275-4537 1548-1811

Vicia faba

endornavirus

VfEV YP_438201 5375-5637 1947-2212

Phytophthora

endornavirus 1*

PEV1 YP_241110 4259-4522

Gremmeniella abietina

type B RNA virus XL*

GaBRV-XL YP_529670 3052-3308

Tymoviridae

Tymovirus Turnip yellow mosaic

virus

TYMV NP_663297 1478-1729 958-1207

Marafivirus maize rayado fino

virus MRFV NP_734076 218-468

Maculavirus Grapevine fleck virus GFkV NP_542612 1588-1837 1050-1279

Flexiviridae

Potexvirus Potato virus X PVX NP_040882 1140-1397 717-965

Allexivirus Shallot virus X ShVX NP_620648 1319-1569 910-1144

Capillovirus Apple stem grooving

virus

ASGV NP_044335 1262-1517

Carlavirus Potato virus M PVM NP_705707 183-436

Foveavirus Apple stem pitting

virus

ASPV NP_604464 1867-2117 1356-1635

Mandarivirus Indian citrus ringspot

virus

ICRSV NP_203553 1341-1598

Trichovirus Apple chlorotic leaf

spot virus

ACLSV NP_040551 1539-1789 1040-1307

Vitivirus Grapevine virus A GVA NP_619662 1375-1625

Luteoviridae

Luteovirus Soybean dwarf virus SbDV NP_150431 484-757

Umbravirus

Tobacco bushy top

virus TBTV NP_733848 124-401

Benyvirus

Beet necrotic yellow

vein virus-F2, France

BNYVV-B-Eur CAA28795 1737-1997 927-1182

Beet soil-borne mosaic

virus BSBMV NP_612601 1745-2005 929-1184

Coronaviridae

Coronavirus Infectious bronchitis

virus

IBV NP_066134 5132-5447

Unassigned Mycovirus

Sclerotinia

sclerotiorum

SsDRV YP_325662 1273-1530

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debilitation-associated

RNA virus

Sclerotinia

sclerotiorum RNA

virus-L

SsRV-L ACE88957 1275-1530 640-878

2

* Tentative members in the designated genera are indicated by asterisks and the names are not 3

italicized 4

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5

Table 2: Percent amino acid sequence identity and similarity between the methyltransferase, 6

helicase and RdRp motifs of SsRV-L and those of other selected alphavirus-like positive-strand 7

RNA viruses. 8

Genus Virus

Abbreviat

ion Accession no. Identity Similarity

Refer

ence

Hepevirus Avian hepatitis E virus* AHEV AAS45830 63/241 (26%) 93/241 (38%) 23

Tobamovirus Tomato mosaic virus ToMV CAD10425 57/240 (23%) 97/240 (40%) Un-pub

lished

Tobacco mosaic virus TMV AAD44327 57/240 (23%) 97/240 (40%) 56

Pepper mild mottle virus PMMoV CAC59955 56/237 (23%) 98/237 (41%) 63

Cucumber green mottle

mosaic virus CGMMV BAA87620 54/217 (24%) 92/217 (42%) 61

Rehmannia mosaic virus* ReMV YP_001041889 56/240 (23%) 95/240 (39%) 68

Meth

yltra

nsfera

se

Betatetravir

us

Nudaurelia capensis beta

virus NβV NP_048059 63/256 (24%) 99/256 (38%) 16

Hepevirus Avian hepatitis E virus* AHEV AAS45830 67/228 (29%) 94/228 (41%) 23

Hepatitis E virus JRA1 HEV-JRA

1 BAB93536 66/229 (28%) 96/229 (41%) 60

Swine hepatitis E virus HEV-

swine ABB88699 82/284 (28%) 122/284 (42%)

Un-pub

lished

Omegatetrav

irus

Helicoverpa armigera

stunt virus HaSV NP_049235 68/240 (28%) 104/240 (43%) 15

RN

A h

elicase

Dendrolimus punctatus

tetravirus* DpTV YP_025094 66/241 (27%) 106/241 (43%) 64

Hepevirus Hepatitis E virus Yam67 HEV-Yam

67 AAM66329 68/250 (27%) 108/250 (43%) 24

Swine hepatitis E virus HEV-

swine ABB88699 70/245 (28%) 108/245 (44%)

Un-pub

lished

Closteroviru

s Beet yellows virus BYV AAC25115 79/335 (23%) 130/335 (38%) 45

Mint virus 1* MV1 YP_224091 65/276 (23%) 111/276 (40%) 62

Grapevine

leafroll-associated virus 2 GLRAV-2 AAC40856 65/280 (23%) 110/280 (39%) 69

Rd

RP

Grapevine rootstock stem GRSLAV NP_835337 66/280 (23%) 112/280 (40%) Un-pub

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lesion associated virus* lished

Citrus tristeza virus CTV AAO12717 70/284 (24%) 112/284 (39%) 53

Unassigned

Closteroviri

dae

Mint vein banding virus* MVBV AAS57939 92/384 (23%) 152/384 (39%) 62

Ampelovirus Plum bark necrosis and

stem pitting-associated

virus*

PBNSTaV YP_001552324 84/346 (24%) 144/346 (41%) 2

Little cherry virus 2 LChV-2 AAP87784 75/308 (24%) 124/308 (40%) 50

9

* Tentaive members in the designated genera are indicated by asterisks and the names are not 10

italicized 11

The number of identical or similar amino acid residues/total number of residues included in the 12

sequence comparison analysis is shown. Values in parentheses are the identity or similarity scores 13

as calculated by the BLAST program 14

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